Previous data have shown a radiation-induced hyporesponsiveness of Vasoactive Intestinal Peptide (VIP)-stimulated short circuit current responses (Isc) in rat distal colon. This finding was associated with reduced cAMP accumulation in isolated crypts and decreased adenylyl cyclase (AC) activity. Such alterations may involve inhibition of the cAMP pathway by [Ca2+]i, cGMP or altered expression of AC isoforms. The present study has addressed such possible mechanisms using co-stimulation studies in addition to determination of the frequency and localization of the AC isoforms present in colonic mucosa. Animals were exposed to a single abdominal irradiation (9 Gy) under gaseous anaesthesia (isoflurane 2.5%, 0.4 L /min) and studied 4 days after exposure. After euthanasia (overdose of sodium pentobarbitone), samples of distal colon were used for (a) Isc determinations in Ussing chambers and (b) full-thickness tissue for localisation of 8 AC isoforms by immunohistochemistry. All experiments were conducted according to the French regulations for animal experimentation (Ministry of the Agriculture Act No. 87848, October 19, 1987). In control animals, VIP (1microM) and carbachol (50microM) in combination increased delta Isc responses to 143+/-32 microA/cm2 (n=5) which was higher than VIP alone (94+/-15 microA/cm2) but not significantly different from carbachol alone (161+/-15 microA/cm2). Addition of SNP (100 microM) slightly reduced the VIP response (76+/-25 microA/cm2). Irradiation reduced VIP-stimulated Isc responses but carbachol- or VIP+carbachol- stimulated responses were unchanged. VIP and SNP co-stimulated delta Isc, responses however were reduced by 41% (P<0.05 Student's t test, n=5). Addition of an iNOS inhibitor (L-NIL) did not markedly restore this hyposensitivity. For the first time, localisation of most of the AC isoforms bas been demonstrated in rat distal colon. Only isoforms III (up-regulated by Ca2+), IV and V/VI (down-regulated by Ca2+ and NO) were present in crypts. Isoform staining was greatest at the surface and declined toward the crypt base. After irradiation, only the upper part of the crypt remained stained without changes of the predominant isoforms. In conclusion, these data show that alteration of VIP-induced Isc responses by [Ca2+]i-mediated pathways is unlikely in both control and irradiated groups. However NO appears to exacerbate the radiation-induced hyporesponsiveness to VIP which is in agreement with the presence of isoform V/VI in crypts. Thus, alteration of the cAMP pathway may be, in part, due to an inhibitory effect of NO perhaps via cGMP. Furthermore, reduced numbers of AC units are in accordance with the lower VIP responses seen 4 days after irradiation.